Polymer-grafted nanobins

ABSTRACT

Provided herein are small unilaminar vesicles with surface-displayed polymer moieties, and methods of use and manufacture thereof. In particular, provided herein are polymer-grafted nanobins, and methods of drug delivery therewith.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the priority benefit of U.S. ProvisionalPatent Application 62/110,055, filed Jan. 30, 2015, which isincorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This is invention was made with government support under P30 CA060553,U01 CA151461 and U54 CA151880 awarded by the National Institutes ofHealth; and FA9550-11-1-0275 awarded by the Air Force Office ofScientific Research. The government has certain rights in the invention.

FIELD

Provided herein are unilaminar vesicles with surface-displayed polymermoieties, and methods of use and manufacture thereof. In particular,provided herein are polymer-grafted nanobins, and methods of drugdelivery therewith.

BACKGROUND

The hydrodynamic diameter (DH) of a nanoparticle strongly influences itsbehavior in vivo. A series of recent reports indicate that particleswith DH of 15-50 nm may be ideal for various biomedical applicationsincluding delivery of therapeutics to hypovascularized cancers (e.g.,pancreatic cancer) (Cabral et al. Nat. Nanotechnol. 2011, 6 (12),815-823; herein incorporated by reference in its entirety), mediatingtherapeutic transport across the blood-brain barrier (Fresta et al.Pharm. Res. 1995, 12 (11), 1769-1774; herein incorporated by referencein its entirety), serving as carriers for transdermal medicine (Sonavaneet al. Colloids Surf. B 2008, 65 (1), 1-10.: herein incorporated byreference in its entirety), and selectively targeting lymph nodes forvaccination (Reddy et al. Nat. Biotechnol. 2007, 25 (10), 1159-1164.).Particles smaller than 15 nm are rapidly excreted via kidney filtration(Choi et al. Nat. Biotechnol. 2007, 25 (10), 1165-1170; hereinincorporated by reference in its entirety), whereas particles largerthan 50 nm accumulate in organs (De Jong et al. Biomaterials 2008, 29(12), 1912-1919;herein incorporated by reference in its entirety) andpoorly penetrate hypovascularized tissues (Cabral et al. Nat.Nanotechnol. 2011, 6 (12), 815-823; herein incorporated by reference inits entirety). Thus, a major direction in current biodelivery researchis the development of stable, biocompatible nanoparticles in the 15-50nm size range that can be used in the tissue-specific delivery of atherapeutic payload.

The liposome, a self-assembled, spherical lipid bilayer, is arguably theperfect bio-delivery system due to its inherent biocompatibility,tunable size, robust and easy preparation, ability to encapsulate atherapeutic payload, and proven clinical success as a delivery vehiclefor small-molecule drugs (Torchilin. Nat. Rev. Drug Discovery 2005, 4(2), 145-160;herein incorporated by reference in its entirety). Thusfar, most liposome-based systems investigated for biodelivery havediameters greater than 100 nm, even though small unilamellar vesicles(SUVs), which are liposomes in the 15-50 nm size range (Silva &Cavaco-Paulo. Biomacromolecules 2011, 12 (10), 3353-3368; hereinincorporated by reference in its entirety), can easily be generated bysonicating larger liposome vesicles. This dearth of applications forSUVs stems from their poor colloid stability, high fusogenicity, anduncontrolled payload leakage. All of these limitations can be attributedto the highly strained membrane curvature of SUVs and a large level ofmolecular disorder in their lipid bilayers, which can induceinter-particle fusion and lead to flocculation during storage (Lin etal. Langmuir 2012, 28 (1), 689-700; herein incorporated by reference inits entirety). The rapid fusion of SUVs has enabled their use as a modelsystem for biomembrane research (Franzin & Macdonald. Biochemistry 1997,36 (9), 2360-2370; herein incorporated by reference in its entirety),but strongly hindered their application in biodelivery. Indeed, due totheir poor colloid stabilities and inherent leakage of encapsulatedcargo (Mcconnell & Schullery. Biochim. Biophys. Acta 1985, 818 (1),13-22; herein incorporated by reference in its entirety), theseparticles have only been employed as a carrier for in vivo therapeuticdelivery in a handful of cases over the last three decades (Forssen etal. Cancer Res. 1992, 52 (12), 3255-3261; Fresta et al. Pharm. Res.1995, 12 (11), 1769-1774; herein incorporated by reference in theirentireties). While a few strategies for overcoming the limitations ofSUVs have been investigated, most have limited success. For example, theincorporation of a negatively charged lipid into SUVs reduced theleakage of an encapsulated fluorescent dye (Mercadal et al. Biochim.Biophys. Acta, Biomembr. 1995, 1235 (2), 281-288; herein incorporated byreference in its entirety), but did not confer long-term stability. Theonly reported example of stabilization of fusogenic SUVs is by Wunderand coworkers, who employed Si0₂ nanoparticles as a surface coating thatinhibits vesicle aggregation (Savarala et al. ACS Nano 2011, 5 (4),2619-2628; herein incorporated by reference in its entirety). However,these SiO₂-stabilized SUVs readily aggregate in the presence of mMconcentrations of sodium chloride, which would limit their applicationas delivery agents.

SUMMARY

Provided herein are unilaminar vesicles with surface-displayed polymermoieties, and methods of use and manufacture thereof. In particular,provided herein are polymer-grafted nanobins, and methods of drugdelivery therewith. In some embodiments, the polymer-grafted nanobinscomprise small phospholipid-bilayer-based vesicles with lipid-terminatedpolymers embedded in and extending from the bilayer, and methods of drugdelivery therewith.

In some embodiments, provided herein are compositions comprising apolymer-grafted nanobin (PGN), wherein said PGN comprises a smallunilaminer vesicle with surface-exposed polymers extending thereform. Insome embodiments, the small unilaminer vesicle is between 15 and 50 nmin diameter (e.g., 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50nm and ranges therein). In some embodiments, the small unilaminervesicle comprises a phospholipid-containing bilayer. In someembodiments, the small unilaminer vesicle comprises1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),Dipalmitoylphosphatidylcholine (DPPC), and/or1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments,the surface-exposed polymers comprise poly(acrylic acid). In someembodiments, the poly(acrylic acid) extends from cholesterol-terminatedpoly(acrylic acid) (Chol-PAA), wherein the cholesterol portion of theChol-PAA is inserted into the bilayer. In some embodiments, the PGNfurther comprises a molecular payload encapsulated within said smallunilaminar vesicle. In some embodiments, the molecular payload comprisesa small molecule, peptide, or nucleic acid. In some embodiments, the PGNreleases 20% or less of said payload over the course of one month atnormal physiologic conditions (e.g., 20%, 15%, 10%, 5%, 1%, or less, andranges therein). In some embodiments, the PGN releases 0.1 to 20% ofsaid payload over the course of one month at normal physiologicconditions (e.g., 0.1% to 5%, 0.5% to 3%, 1% to 5%, 2% to 15%, 3% to20%, or other combinations of range endpoints therein). In someembodiments, the PGN releases at least 50% (e.g., 50%, 60%, 70%, 80%,85%, 90%, 95%, 99%, and ranges therein) of said payload over the courseof less than one hour at a pH between 4.0 and 6.0. In some embodiments,the PGN releases between 50% and 99% (e.g., 50% to 70%, 60% to 95%, 70%to 85%, 80% to 90%, 85% to 99%, 90% to 99%, 75% to 95%, or othercombinations of range endpoints therein) of said payload over the courseof less than one hour at a pH between 4.0 and 6.0. In some embodiments,the PGN releases at least 50% (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%,99%, and ranges therein) of said payload over the course of less thanone hour at a pH below 6.0 (e.g., pH 5.8, pH 5.6, pH 5.4, pH 5.2, pH5.0, pH 4.8, pH 4.6, pH 4.4, pH 4.2, pH 4.0, or lower, and rangestherein). In some embodiments, the surface-exposed polymers are notcross-linked.

In some embodiments, provided herein are compositions comprising apolymer-grafted nanobin (PGN), wherein said PGN comprises a smallphospholipid-based unilaminer vesicle (SUV) comprisingcholesterol-terminated poly(acrylic acid) groups (Chol-PAA), whereinsaid Chol-PAA is oriented such that said poly(acrylic acid) is surfaceexposed and extends from said SUV into the surrounding environment,wherein said SUV encompasses a molecular payload comprising one or morepeptides, nucleic acids, and/or small molecules.

In some embodiments, provided herein are methods of drug delivery to alow-pH microenvironment within a subject or cell comprising: (a)administering a polymer-grafted nanobin (PGN) to said subject or cell,wherein said PGN comprises a small phospholipid-based unilaminer vesicle(SUV) comprising cholesterol-terminated poly(acrylic acid) groups(Chol-PAA), wherein said Chol-PAA is oriented such that saidpoly(acrylic acid) is surface exposed and extends from said SUV into thesurrounding environment, wherein said SUV encompasses a molecularpayload comprising one or more therapeutic agents; and (b) allowing saidPGN to migrate from physiologic conditions to an acidicmicroenvironment. In some embodiments, the PGN is administered locallyat or near the acidic microenvironment. In some embodiments, the PGN isadministered systemically. In some embodiments, the acidicmicroenvironment is a tumor. In some embodiments, the therapeutic agentis a chemotherapeutic. In some embodiments, payload is a nucleicacid-based therapy (e.g., siRNA, antisense RNA, miRNA, ribozyme, vectorencoding a gene, etc.). In some embodiments, the PGN releases less than20% of said payload over the course of one month at normal physiologicconditions and releases at least 50% of said payload over the course ofless than one hour at a pH between 4.0 and 6.0. In some embodiments, thePGN is co-administered with an additional therapeutic agent. In someembodiments, methods further comprise a step of surgically removing thetumor (e.g., the administration step is performed before, during, orafter surgical removal of the tumor). In some embodiments, methodsfurther comprise a step of radiating or ablating the tumor (e.g., theadministration step is performed before, during, or after radiating orablating the tumor).

In some embodiments, provided herein are methods of triggering therelease of a molecular payload comprising: (a) encapsulating themolecular payload within a polymer-grafted nanobin (PGN); (b) placingthe PGN under approximately physiologic conditions; and (c) triggeringthe release of the molecular payload by lowering the pH to below 6.0. Insome embodiments, the PGN comprises a small phospholipid-basedunilaminer vesicle (SUV) comprising cholesterol-terminated poly(acrylicacid) groups (Chol-PAA), wherein said Chol-PAA is oriented such thatsaid poly(acrylic acid) is surface exposed and extends from said SUVinto the surrounding environment. In some embodiments, release istriggered by lowering the pH below 5.5. In some embodiments, loweringthe pH comprises allowing the PNG to migrate into a low pH environment.In some embodiments, lowering the pH comprises adding a reagent to lowerthe pH of the PGN's environment.

In some embodiments, provided herein are polymer-grafted nanobin(PGN)-encapsulated therapeutics for use as medicaments. In someembodiments, provided herein is the use of a polymer-grafted nanobin(PGN)-encapsulated therapeutic for the treatment of a disease orcondition. In some embodiments, provided herein is the use of a PGN forthe manufacture of a medicament for a therapeutic application.

In some embodiments, provided herein are methods of preparing apolymer-grafted nanobin (PGN) comprising: (a) preparing a lipid mixtureby dissolving selected lipids in an organic solvent; (b) hydrating theproduct of step (a) with an aqueous hydration solvent to form liposomes;(c) sizing the liposomes to yield small unilaminar vesicles (SUVs); and(d) incubating the SUVs with lipid-anchored polymer to generate PGNswith surface exposed polymer. In some embodiments, the sizing stepcomprises one or more of: (i) high sheer mixing, (ii) extruding throughone or more filters, and (iii) sonicating. In some embodiments, methodsfurther comprise a step of removing the organic solvent (e.g., prior tostep (b), after step (b), etc.). In some embodiments, the lipid mixturecomprises phospholipids. In some embodiments, lipid mixture comprises atleast 70% phosphiolipids (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 99%,ranges therein). In some embodiments, the lipid mixture comprisesdimyristoyl-sn-glycero-3-phosphocholine (DMPC),Dipalmitoylphosphatidylcholine (DPPC), and/or1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments,sizing results in SUVs between 15 and 50 nm in diameter (e.g., 15 nm, 20nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, ranges therein). In someembodiments, sizing results in SUVs with a PDI of less than 0.3 (e.g.,0.25, 0.2, 0.15, 0.1, 0.05, or less, and ranges therein). In someembodiments, the lipid constituent of the lipid-anchored polymer is asterol. In some embodiments, the sterol is cholesterol. In someembodiments, the polymer constituent of the lipid-anchored polymer ispoly(acrylic acid). In some embodiments, the lipid-anchored polymer ischolesterol-terminated poly(acrylic acid) (Chol-PAA). In someembodiments, methods further comprise a step of hydrating withhydrophilic molecular agent to yield an encapsulated molecular payload.In some embodiments, the step of hydrating with hydrophilic molecularagent is performed between steps (b) and (c). In some embodiments, thestep of hydrating with hydrophilic molecular agent is performed betweensteps (c) and (d).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A schematic illustration of the drop-in synthesis of PGN: abatch of preformed liposomes are sonicated into SUVs using a probesonicator, separated from heavy impurities by ultracentrifugation, andstabilized by the addition of Chol-PAA.

FIG. 2. Comparative stability and cargo-retention properties of SUVs andPGNs derived from DOPC, DMPC, and DPPC. (A-C) Hydrodynamic diameters(DH) of three independent batches of SUVs and PGNs, as monitored bydynamic light scattering (DLS) over sixteen weeks. The shown DH valuesare the volume-average distribution data for the most significantpopulation, which emphasizes the presence of aggregation compared todata derived based on a number distribution. The error bars representthe standard deviations between three different batches. (D-F) The meancalcein leakage profiles over a six-week period for three batches ofSUVs and PGNs. The error bars represent the average standard deviationbetween three different batches.

FIG. 3. (A) TE micrograph and photographic image (inset) of DPPC-SUVsthree months after synthesis, illustrating the instability of thisformulation. (B) TE micrograph and photographic image (inset) ofDPPC-PGNs three months after synthesis, illustrating the exceptionalstability of this formulation.

FIG. 4. The release of DXR, as measured by fluorescence, from DOPC-PGNand SUVs in response to in-situ acidification of pH 7.4 to pH 5.0.

FIG. 5. A schematic illustration of the proposed mechanism by whichclustering of the grafted Chol-PAA chains on the surface of SUV-derivedPGNs creates large defects on the already highly curved membranes,leading to spontaneous release of the payload.

FIG. 6. The arithmetic mean DH for PEG2000-, PEG3000-, andPEG600-grafted DPPC-based SUVs over eight weeks of storage, asquantified by DLS.

FIG. 7. Photographic image of PEG-SUVs after 3 months of storage at 4°C.: Chol-PEG₆₀₀ modified DPPC (left), DPPC-PEG₂₀₀₀ (middle), andDPPC-PEG₃₀₀₀ (right).

FIG. 8. Photographic images of SUV and PGN samples after six months ofstorage at 4° C. (A) DPPC-PGN (left), DPPC-SUVs (right). (B) DMPC-PGN(left), DMPC-SUVs (right). (C) DOPC-PGN (left), DOPC-SUVs (right). (D)DPPC-SUVs with 5 (left), 10 (left middle), 15 (right middle), and 20(right) mol % cholesterol addition.

FIG. 9. TEM images of a sample of PEG₂₀₀₀-grafted DPPC-SUVs stored at 4°C. two days after preparation (A) and two months after preparation (B).

FIG. 10. TEM images of a sample of DPPC-SUVs stored at 4° C. two daysafter preparation (A) and six months after preparation (B).

FIG. 11. TEM images of a sample of DPPC-PGN stored at 4° C. two daysafter preparation (A) and six months after preparation (B).

DEFINITIONS

As used herein, the term “lipid” refers to a variety of compounds thatare characterized by their solubility in organic solvents. Suchcompounds include, but are not limited to, fats, waxes, steroids,sterols, glycolipids, glycosphingolipids (including gangliosides),phospholipids, terpenes, fat-soluble vitamins, prostaglandins,carotenes, and chlorophylls. As used herein, the terms “lipid-basedmaterials” and “lipid assemblies” refers to any material that containslipids. In some embodiments, “lipid assemblies” are structuresincluding, but not limited to vesicles, liposomes, films, micelles,dendrimers, monolayers, bilayers, tubules, rods, and coils.

As used herein, the term “vesicle” refers to a small,membrane-bilayer-enclosed structure. Membranes of vesicles may compriselipids, proteins, glycolipids, steroids or other components associatedwith membranes. Vesicles can be naturally generated (e.g., the vesiclespresent in the cytoplasm of cells that transport molecules and partitionspecific cellular functions) or can be synthetic (e.g., liposomes).

As used herein, the term “liposome” refers to artificially-producedspherical lipid complexes that are induced to segregate out of aqueousmedia. Liposomes are composed of amphiphilic lipids arranged in aspherical bilayer. Liposomes are unilamellar (e.g., contained within asingle bilayer of amphiphilic components (e.g., lipids)) ormultilamellar vesicles.

As used herein, the term “surface exposed” refers to molecules (e.g.,polymers) that are present at the surface of a structure (e.g., a lipidassembly) and are accessible to the solvent surrounding the structure aswell as being accessible to other agents contained in the solvent. Theterms “payload” and “molecular payload” refer to any chemical entity,pharmaceutical, drug (such drug can be, but not limited to, a smallmolecule, an inorganic solid, a polymer, or a biopolymer), smallmolecule, nucleic acid (e.g., DNA, RNA, siRNA, etc.), protein, peptideand the like that is encompassed within a liposomal or vesicularformulation described herein. A “therapeutic payload” refers to apayload with the intended effect of providing a subject with treatmentor prevention of a disease or condition.

As used herein, the term “nanobin” refers to a nanoscale moleculardelivery vehicle wherein a molecular payload is encapsulated with alipid bilayer and polymeric exterior. Exemplary nanobins are less than100 nm in diameter, have an aqueous core within a lipid bilayer forencapsulation of hydrophilic payloads (e.g., small molecules, nucleicacids, peptides etc.), and have surface accessible polymers anchored tothe bilayer by a conjugated lipid components (e.g.,cholesterol-terminated poly(acrylic acid), etc.).

As used herein, the term “physiologic conditions” refers to solution orreaction conditions roughly simulating those most commonly found inmammalian organisms, particularly humans (e.g., not relating to specificmicorenvironments within organisms (e.g., not the acidic conditions (pH5.0) commonly found in tumor microenvironments and cellular lateendosomes) or other rare conditions). While variables such astemperature, availability of cations, and pH ranges may vary,“physiologic conditions” typically mean a temperature of 35-40° C., withabout 37° C. being particularly preferred, and a pH of 7.0-8.0, withabout 7.5 being particularly preferred. The conditions may also includethe availability of cations, preferably divalent and/or monovalentcations, with a concentration of about 2-15 mM Mg²⁺ and 0 1.0 M Na⁺being particularly preferred.

DETAILED DESCRIPTION

Provided herein are unilaminar vesicles with surface-displayed polymermoieties, and methods of use and manufacture thereof. In particular,provided herein are polymer-grafted nanobins, and methods of drugdelivery therewith. In some embodiments, the polymer-grafted nanobinscomprise small phospholipid-bilayer-based vesicles with lipid-terminatedpolymers embedded in and extending from the bilayer, and methods of drugdelivery therewith.

In some embodiments, provided herein are fusogenic SUVs and experimentsdemonstrating their long-term stabilization in biologically relevantmedia (pH 7.4, 150 mM NaCl). By utilizing polymer surface grafts (e.g.,short poly(acrylic acid) (PAA) surface grafts), metastable cargo-loadedSUVs were converted into highly stable polymer-grafted nanobins (PGNs).Typically, the potentials of these PGNs are quite negative (−45±3 mV,Table 5), which is predicted by the Derjaguin, Landau, Verwey andOverbeek (DLVO) theory to be in the right repulsive range for theobserved stabilization (Laaksonen et al. ChemPhysChem 2006, 7 (10),2143-2149; herein incorporated by reference in its entirety). Not onlycan the polymer (e.g., PAA) surface grafts stabilize the PGNs for oversix months with minimal cargo leakage, these grafts also cause PGNs tospontaneously release their cargos under the acidic conditions (pH 5.0)commonly found in tumor microenvironments, cellular late endosomes, andother acidic environemnts (Lee et al. ACS Nano 2011, 5 (5), 3961-3969;herein incorporated by reference in its entirety). This switchablecombination of properties makes PGNs a class of “smart” nanocarriers forbiological applications.

Experiments conducted during development of embodiments described hereindemonstrate that biocompatible Chol-PAA polymer-grafts endow metastableSUVs with a high potential that enables them to remain dispersed inbiologically relevant solutions for long periods. Additionally, theCholPAA-grafted PGNs exhibit a combination of properties that are highlydesirable for smart nanocarriers, including: remarkable cargo retentionunder physiologically relevant conditions along with a facile, abruptrelease of the payload in response to acidification. As Chol-PAAgrafting endows a large number of carboxyl groups (˜20,000/particle) onthe PGN surface, this simple drop-in modification extends the utility ofSUVs beyond the immediate benefits of their small sizes. Suchfunctionalization can be used as a robust conjugation handle for otherfunctional groups such as cellular-targeting ligands (Lee et al. J. Am.Chem. Soc. 2009, 131 (26), 9311-9320; herein incorporated by referencein its entirety), therapeutics (Lee et al. J. Am. Chem. Soc. 2010, 132(48), 17130-17138; herein incorporated by reference in its entirety),and imaging agents (Lee et al. Angew. Chem., Int. Ed. 2010, 49 (51),9960-9964; herein incorporated by reference in its entirety). Together,the desirable combination of high stabilities, triggered releasecapability, and functionalization handles enable PGN as a robust andversatile scaffold for the smart and efficient delivery of cargos forboth targeted biological and non-biological applications.

In some embodiments, the liposomes and/or vesicles (e.g., SUVs, PGNs,etc.) described herein are composed of any suitable lipids,phospholipids, steroids (e.g., sterols), and other components useful orsuitable for the formation of such structures. For example, suitablephospholipds for the formation of liposomes include:1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),Dipalmitoylphosphatidylcholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-Bis(dimethylphosphino)ethane (DMPE),1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE),1,2-ditetradecanoyl-sn-glycero-3-phosphate (DMPA),1,2-Dipalmitoyl-sn-glycero-3-phosphate (DPPA),1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphate (DOPA),1,2-Dimyristoyl-sn-Glycero-3-PhosphoGlycerol (DMPG),1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS),1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS),1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS),1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine (DSPE),1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), etc. Similarly,suitable sterols for use in the formation of liposomes and othervesicles described herein include, but are not limited to: cholesterol,ergosterol, hopanoids, phytosterol, stanol, etc. Further, any of theaforementioned components of liposomes and vesicles may be appropriatelymodified (e.g., terminally modified) with moieties, e.g., forinteraction with the solvent surrounding the structure or componentnstherein. For example, one or more liposome or vesicle components may beterminally modified, with a suitable moiety such as: poly(ethyleneglycol) (PEG), poly(ethylene oxide)diacrylate (PEODA), polyacrylic acid,poly vinyl alcohol, collagen, poly(D,L-lactide-co-glycolide (PLGA),polyglactin, alginate, polyglycolic acid (PGA), other polyesters (e.g.,poly-(L-lactic acid) (PLLA), polyanhydrides, poly(diol citrate)s, etc.),etc. Examples of polymer modified lipids include cholesterol-terminatedpoly(acrylic acid) (Chol-PAA) and poly(ethylene glycol) modified DSPE(e.g., PE-PEG600, PE-PEG2000, PE-PEG3000, etc.), poly(ethylene glycol)modified cholesterol (Chol-PEG), etc.

In some embodiments, PGNs and other liposomal and/or vesicularformulations described herein comprise between 70 mol % and 100 mol %phospholipid content (e.g., 70 mol %, 75 mol %, 80 mol %, 85 mol %, 90mol %, 95 mol %, 99 mol %, and ranges therein (e.g., 85-95 mol %) withinthe lipid bilayer. In some embodiments, a single type of phospholipid ispresent (e.g., DPPC, DMPC, DOPC, etc.). In some embodiments, multipletypes of the phospholipids described herein make up the bilayer. In someembodiments, lipid-terminated polymer (e.g., Chol-PAA) comprises 1-30mol % of the content of the lipid bilayer (e.g., 1 mol %, 2 mol %, 5 mol%, 10 mol %, 15 mol %, 20 mol %, 25 mol %, 30 mol %, and ranges therein(e.g., 5-20 mol %)).

In some embodiments, PGN and/or other liposomal/vesicular formulationsfurther comprise a cryo- and/or lyo-protecting agent. During storage ofliposomes the phospholipids may undergo hydrolysis. One simple way ofpreventing decomposition of the phospholipids in the liposomeformulation is by freezing or freeze-drying. Freezing may however induceleakage of the liposome formulation and result in release of theencapsulated drug. Addition of a cryo-protecting agent may prevent orreduce leakage from a preparation after freezing. Examples of agentsthat may be used as cryo-protecting agents may without limitation bedisaccharides such as sucrose, maltose and/or trehalose. Such agents maybe used at various concentrations depending on the preparation and theselected agent such as to obtain an isotonic solution. In someembodiments, PGN and/or other liposomal/vesicular formulations arefreeze-dried, stored and the reconstituted such that a substantialportion of the internal contents are retained. Dehydration generallyrequires use of a lyo-protecting agent such as a disaccharide (sucrose,maltose or trehalose) at both the inside and outside interfaces of thebilayer. This hydrophilic compound prevents the rearrangement of thelipids in the formulation, so that the size and contents are maintainedduring the drying procedure and through subsequent reconstitution.Appropriate qualities for such drying protecting agents are that theypossess stereo chemical features that preserve the intermolecularspacing of the liposome bilayer components.

In some embodiments, PGNs comprise one or more functional surfacemoieties to confer one or more beneficial functionalities to the PGNs.Exemplary functional moieties may include, but are not limited to: adetectable moiety (e.g., fluorophore, chromophore, contrast agent,radionuclide, etc.), a targeting/binding/interaction moiety (e.g.,antibody, antibody fragment, binding peptide (e.g., recognized by a cellsurface receptor), etc.), etc. For example, suitabale functionalmoieties may include: one or more small molecules (e.g., drugs,drug-like molecules), biomolecules, a peptide or polypeptide (protein)including an antibody or a fragment thereof, a His-tag, a FLAG tag, aStrep-tag, an enzyme, a cofactor, a coenzyme, a substrate for an enzyme,a suicide substrate, a receptor, double stranded or single strandednucleic acid (e.g., RNA or DNA), e.g., capable of binding a protein, aglycoprotein, a polysaccharide, a peptide-nucleic acid (PNA), a solidsupport (e.g., a sedimental particle such as a magnetic particle, asepharose or cellulose bead, a membrane, a glass slide, cellulose,alginate, plastic or other synthetically prepared polymer (e.g., aneppendorf tube or a well of a multi-well plate, etc.), etc.), a drug(e.g., chemotherapeutic), pH sensor, a radionuclide, a contrast agent, achelating agent, a cross-linking group (e.g., a succinimidyl ester oraldehyde, maleimide, etc.), glutathione, biotin, streptavidin, one ormore dyes (e.g., a xanthene dye, a calcium sensitive dye (e.g.,1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxy-9-xanthenyl)-phenoxy]-2-(2′-am--ino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (Fluo-3), etc.),a sodium sensitive dye (e.g., 1,3-benzenedicarboxylic acid,4,4′-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis(PBFI), etc.), a NO sensitive dye (e.g.,4-amino-5-methylamino-2′,7′-difluorescein), or other fluorophore, ahapten or an immunogenic molecule (e.g., one which is bound byantibodies specific for that molecule), etc.

Functional moieties may be attached to the polymer portion of thelipid-terminated polymer components of PGNs (e.g., the poly(acrylicacid) portion of Chol-PAA), may be attached to the head group ofphospholipids within the bilayer, may be attached to lipophilic moietiesembedded within the bilayer (e.g., cholesterol groups), etc. Functionalmoieties may be directly attached to components of the PGN or may beconnected by a suitable linker (e.g., carbon-containing chain, peptide,cleavable linker, etc.).

In some embodiments, PGN's comprise one or more functional moieties todirect and/or localize the PGNs to intended locations of drug delivery(e.g., acidic tumor microenvironments). In some embodiments, PGNsdisplay a tumor-targeting ligand to direct the PGNs to tumormicroenvironments, such tumor-targeting ligands areselected from folicacid, retinoic acid, a peptide, an estrogen analog, transferrin, andgranulocyte-macrophage colony stimulating factor. In some embodiments,the targeting moiety comprises an antibody. In some embodiments, theantibody is selected from RITUXAN, HERCEPTIN, CAMPATH-1H, HM1.24,anti-HER2, Anti-CD38, HuM195, HP67.6, TRAIL mAb, transferin, ATN-291,and prolactin. Any moiety capable of directing a PGN to a tumor may finduse in emboidments herein.

In some embodiments, provided herein are methods of generating orsynthesizing liposomes and/or vesicles from component elements. Suitabletechniques for assembling such components into liposomes are understoodin the field. For example, an exemplary method comprises the steps of:(a) preparing a lipid mixture by dissolving selected lipids in anorganic solvent; (b) hydrating the product of step (a) with an aqueoushydration solvent so as to form liposomes; and (c) removing the organicsolvent of step (a) either before addition of the aqueous hydrationsolvent or after the addition of the aqueous hydration solvent. Suitablemethods may further comprise steps of, for example, (1) high sheermixing to reduce the size of the liposomes, (2) extruding the liposomesthrough filter(s) to produce liposomes of a certain mean size, and/or(3) sonicating the liposomal formulation to produce liposomes of acertain size.

PGNs, SUVs, and/or other liposomal/vesicular formulations may be loadedwith at least one molecular payload (e.g., therapeutic agent) bysuitable methods understood in the field. For example, by solubilizingthe compound in the organic solvent or hydration solvent used to preparethe liposomes. Alternatively, an ionizable therapeutic agent can beloaded into vesicles by establishing an electrochemical potential, e.g.,by way of a pH gradient, across the outermost bilayer, and then addingthe ionizable therapeutic agent to the aqueous medium external to theliposome.

In some embodiments, methods further comprise a step of changing theexterior aqueous phase of the formulation. In some embodiments, theaqueous phase initially comprises the hydration solvent. The exterioraqueous phase may be changed by centrifugation, ultrafiltration,dialysis or similar in order to prepare a liposomal formulationcomprising vesicles (e.g. PNG) in a solution of defined composition ofthe ex terior aqueous phase. In some embodiments, bioactive compounds(therapeutic agents) are only present inside or attached to the vesicles(e.g. PNG) and not as free compounds in solution. Preferably, the drugencapsulation in the vesicles (e.g. PNG) is >70%, more preferably >95%and most preferably >99%. The degree of drug encapsulation is the ratioof drug encapsulated to the total amount of drug in the formulation.

In some embodiments, the PGN and/or other liposomal/vesicularformulations described herein exhibit hydrodynamic diameters of 15-50 nm(e.g., 15 nm, 20 nm, 15 nm, 20 nm, 15 nm, 20 nm, 15 nm, 20 nm, andranges therein (e.g., 30-50 nm)).

In some embodiments, the Poly Dispersity Index (PDI) of the PGN and/orother liposomal/vesicular formulations described herein do not exceed0.3 (e.g., 0.3, 0.25, 0.2, 0.15, 0.10, 0.05, 0.01, and ranges therein(e.g., 0.05-0.15 PDI)). A PDI value in the ranges described hereinrepresented a relatively narrow particle size-distribution in theformulations. One object of the embodiments described herein is toprovide liposomal and/or vesicular formulations with increased stabilityduring storage. In some embodiments, the PGNs described herein exhibitdispersion and/or degradation of less than 20% of PGNs in a sample(e.g., <20%, <15%, <10%, <5%, <4%, <3%, <2%, <1%, and ranges therein(e.g., 1-5%)) over a time period of at least one month (e.g., >1month, >2 months, >3 months, >6 months, >1 year, and ranges therein(e.g., 6 months to 1 year)).

Liposomes and vesicles can be used to carry various compounds such as,e.g., drugs, encapsulated within the interior aqueous compartment (e.g.,encapsulated within the liposome/vesicle) and/or embedded within thebilayer (e.g., embedded in the liposome/vesicle). Depending on thechemical nature of the compound to be encapsulated it will be localizedto either of the compartments. Currently, there are several parenteralliposome-drug formulations available on the market. Water soluble drugstend to be encapsulated in the aqueous compartment of liposomes, andexamples of drugs encapsulated in liposome's are, e.g., doxorubicin(Doxil), doxorubicin (Myocet) and daunorubicin (DaunoXone). Examples ofdrugs intercalated in the liposome membrane are, e.g., amphotericin B(AmBisome), amphotericin (Albelcet B), benzoporphyrin (Visudyne) andmuramyltripeptide-phosphatidylethanolamine (Junovan). Embodimentsdescribed herein are not limited by the variety of drugs or othermolecular payloads that can be encapsulated within or embedded in thePGN.

Unless specifically stated, the embodiments described herein are not tobe limited by the identity of the potential agents that can be deliveredtherewith. In some embodiments, agents are embedded within the bilayeror linked to hydrophobic moieties within the bilayer but the agent issurface exposed. Due to the triggered release functionality of the PGNsand/or liposomal/vesicular formulations described herein, thecompositions are of particular utility for the encapsulation of watersoluble agents (or solubilized agents) within the PGN (e.g., as amolecular payload) or other formulation and/or delivery/release of thoseagents at a desired time/location. Suitable molecular payloads includesmall molecules (e.g., drugs or drug-like molecules), peptides,polypeptides, nucleic acids (e.g., DNA (e.g., genes, microgenes,DNAzymes, etc.), RNA (e.g., siRNA, miRNA, antisense RNA, RNA decoys,ribozymes), aptamers (e.g., DNA or RNA), nucleic acid vectors (e.g.,encoding genes), etc.), etc. In some embodiments, PGNs are useful forthe delivery of a nucleic acid therapeutic (or other agent) to a desiredcell type or microenvironment (e.g., acidic micorenvironemnt) followedby release of the nucleic acid (or other agent). A description ofnucleic acid therapeutics, for which the delivery systems describedherein may provide utility are described, for example, by Pushpendra etal. in V. A. Erdmann and J. Barciszewski (eds.), From Nucleic AcidsSequences to Molecular Medicine, RNA Technologies, DOI10.1007/978-3-642-27426-8_2, Springer-Verlag Berlin Heidelberg 2012;herein incorporated by reference in its entirety). In some embodiments,the molecular payload comprises a peptide therapeutic; descriptions ofuseful peptide therapeutics include Boohaker et al. Curr Med Chem.2012;19(22):3794-804; Kaspar and Reichert. Drug Discov Today. 2013September;18(17-18):807-17; herein incorporated by reference in theirentireties).

An object of the embodiments described herein is to provide formulationsfor drug delivery that deliver their payload (e.g., a drug) to thetarget site, and release the payload at the target site (e.g., >25%release, >30% release, >40% release, >50% release, >60% release, >70%release, >80% release, >90% release, and ranges therein (e.g., 40-70%))with low uncontrolled/non-specific delivery and/or leakiness (e.g., <1%,<2%, <5%, <10%, <20%, <25%, and ranges therein (e.g., 1-10%)) at anon-target site and/or under non-target conditions (e.g., normalphysiologic conditions).

The PGNs and other liposomal/vesicular formulations provided herein finduse in a variety of applications. As described throughout, PGNs exhibita variety of characteristics that make them particularly well suited fordrug delivery. In particular, due to the pH dependence of their payloadrelease (e.g., little leakage at physiologic pH and bulk release atacidic pH (e.g., <pH 6.0, <pH 5.8, <pH5.6, <pH 5.4, <pH 5.2, or lower),etc.), certain PNGs described herein are particularly well suited fordelivery of a payload (e.g., therapeutic agent) through a physiologicenvironment for release of payload (e.g., delivery of therapeutic) in anacidic environment (e.g., acidic extracellular microenvirnment.Therefore, in some embodiments, PGNs and other liposomal/vesicularformulations provided herein find use in the treatment of cancer (e.g.,solid tumor cancer). In some embodiments, compositions (e.g., comprisingPGNs) described herein find use in drug delivery and the treatment ofcancers, for example, adenomas, carcinomas or sarcomas, and includingbut not limited to: melanoma, brain tumors, neuroblastomas, breastcancer, lung cancer, prostate cancer, cervix cancer, uterine cancer,ovarian cancer, colon cancer, rectum cancer, cancer of the testis,cancer of the kidney, cancer of the liver, cancer of the lip, cancer ofthe tongue, cancer of the stomach, skin cancer, mesotheliomas, bladdercancer, bone tumors, malignant pleural effusions, ascites, meningealcarcinomatosis, head and neck cancers, cancers of endocrine organs suchas: thyroid gland, pituitary gland and suprarenal gland, etc.

As addressed herein, PGNs and/or other liposomal/vesicular formulationsmay display, for example, various targeting moieties that direct thedrug-delivery vehicle to the appropriate tumor and allow release oftherapeutic (e.g., chemotherapeutic) payload in the acidic tumormicroenvironment. In alternative embodiments, PGNs and/or otherliposomal/vesicular formulations are delivered directly to a tumormicroenvironment.

Drug-delivery utilizing PGNs and/or other liposomal/vesicularformulations described herein are not limited to cancer treatment. Forexample, the compositions described herein find use in the delivery toany acidic locations (e.g., vaginal mucosa, stomach, fallopian tubes,etc.).

In some embodiments, because of the ability of the compositionsdescribed herein to stably contain a molecular payload with minimalleakage, such compositions find use in containment of molecular agentsand or bulk release of such agents at a desired time point. Suchfunctionality may be particularly useful in the containment of toxicsubstances and/or the precise timing of the release of a chemicalreactant.

Applications for PGNs and/or other liposomal/vesicular formulations arenot limited to drug-delivery. For example, compositions described hereinmay find use in: the formulation of nutritional/dietary supplements,cosmetics, and lubricants; regenerative medicine; industrialapplications; agricultural applications (e.g., pesticide delivery);veterinary applications; etc.

Experimental EXAMPLE 1 Materials

All lipids—1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) (PE-PEG₂₀₀₀), and2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-3000] (ammonium salt) (PE-PEG₃₀₀₀)—were purchased from AvantiPolar Lipids, Inc., as either a dry powder or a chloroform solution.Cholesterol-PEG₆₀₀,N-tert-butyl-O-[1-[4-(chloromethyl)phenyl]ethyl]-N-(2-methyl-1-phenylpropyl)hydroxylamine,2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide, cholesterol, andtent-butyl acrylate were purchased from Sigma-Aldrich (Milwaukee, Wis.)and used as received. Doxorubicin hydrochloride was purchased fromPolyMed Therapeutics (Houston, Tex.). Ultrapure deionized water (18.2MS) cm resistivity) was obtained from a Millipore system (Billerica,Mass.).

HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-bufferedsaline (HBS) solution (20 mM HEPES, 150 mM NaCl, pH 7.4) was preparedusing standard protocols. Chol-PAA (M_(n)=10.7 kDa and PDI=1.12) wasprepared following a literature procedure (Leevet al. J. Am. Chem. Soc.2007, 129(49), 15096; herein incorporated by reference in its entirety).A 20 mM solution of calcein in HBS was prepared by sonicating theappropriate amount of powdered calcein (Sigma-Aldrich) in HBS for 10minutes using a probe sonicator at room temperature.

EXAMPLE 2 Instrumentation

Phosphorus concentrations of the synthesized materials were determinedusing a Varian Vista MPX (Varian, Inc., Palo Alto, Calif.) simultaneousinductively coupled plasma optical emission spectrometer (ICP-OES).

Polymer molecular weights were determined relative to polystyrenestandards on a Varian PL-GPC 50 Plus (Varian, Inc., Palo Aalto, Calif.)gel-permeation chromatography (GPC) system equipped with Cirrussoftware, a PL-50 RT GPC autosampler, both RI and UV detectors, AgilentResipore guard column, and Agilent Mesopore and Resipore columns (both300×7.5 mm in size) in series. HPLC-grade chloroform was used as aneluent at a flow rate of 1.0 mL/min and the instrument was calibratedusing polystyrene standards (Aldrich Chemical Co., 6 standards,2,330-18,700 Daltons).

Dynamic light scattering (DLS) and zeta potential measurements wereperformed on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK)equipped with a He-Ne laser (633 nm). Non-invasive backscatter method(detection at 173° scattering angle) was used. Correlation data werefitted, using the method of cumulants, to the logarithm of thecorrelation function, yielding the diffusion coefficient (D). Thehydrodynamic diameters (D_(H)) of the small unilamellar vesicles (SUVs)and PCNs were calculated using D and the Stokes-Einstein equation(D_(H)=k_(B)T/3πηD, where k_(B) is the Boltzmann constant, T is theabsolute temperature, and η is the solvent viscosity (η=0.8872 cP forwater)). The polydispersity index (PDI) of liposomes—represented as2c/b², where b and c are first- and second-order coefficients,respectively, in a polynomial of a semi-log correlation function—wascalculated by cumulants analysis. Size distribution of vesicles wasobtained by non-negative least squares (NNLS) analysis.^(S2) Typically,a sizing sample was prepared by adding a small aliquot (10 μL) of thelipid dispersion into a low-volume disposable sizing cuvette (MalvernInstruments, Malvern, UK) filled with 20 mM HBS (750 μL) and thedispersion was briefly mixed with a pipet before data was collected.

Typically, a potential measurement sample was prepared by adding a smallaliquot (20 μL, twice as concentrated as that for the sizing sample toincrease count rate) of the lipid dispersion into a disposable capillarycell (Malvern Instruments, Malvern, UK) filled with 20 mM HBS (740 μL)and the dispersion was briefly mixed with a pipet before data wascollected. The reported data represents the statistical average of thevolume particle size distribution (volume PSD) from five measurementswith 25 scans each, the calculated standard deviation is the errorassociated with each measurement. If two or more significant (>1%),populations occur within one sample, they are reported as relativepercent population (Tables 1-4).

TABLE 1 Volume particle size distribution (volume PSD) data collectedfor three separate batches for each DPPC-, DMPC-, and DOPC-SUVs. If twoor more significant (>1%) populations occur within one sample, they arereported as relative percent populations. Errors are represented asaverage standard deviations of the volume PSD. DPPC-SUV DMPC-SUVDOPC-SUV Time Volume % Volume % Volume % (days) PSD (nm) population PDIPSD (nm) population PDI PSD (nm) population PDI 0 48 ± 3 86 0.57 26 ± 3 100 0.33 32 ± 5 100 0.28 646 ± 35 14 15 58 ± 1 65 0.68 34 ± 2  100 0.4534 ± 3 100 0.25 1450 ± 84  35 30 1450 ± 204 70 1 615 ± 110 88 0.39 35 ±2 100 0.26 52 ± 5 30 28 ± 3  11 60  920 ± 150 100 1 545 ± 230 100 0.3339 ± 2 100 0.31 90 1330 ± 200 100 0.89 654 ± 200 100 0.47 42 ± 1 1000.35 120 1330 ± 250 100 1 715 ± 250 100 0.55 35 ± 2 100 0.31 150 1330 ±200 100 1 740 ± 230 100 0.54 46 ± 6 100 0.35 180 1330 ± 250 100 1 720 ±250 100 0.55 36 ± 5 100 0.32

TABLE 2 Volume particle size distribution (volume PSD) data collectedfor three separate batches for each DPPC-, DMPC-, and DOPC-PGNs. If twoor more significant (>1%) populations occur within one sample, they arereported as a relative percent populations. Error is represented as ±average standard deviations of the volume PSD. DPPC-PGN DMPC-PGNDOPC-PGN Volume Volume Volume Time PSD % PSD % PSD % (days) (nm)population PDI (nm) population PDI (nm) population PDI 0 46 ± 1 100 0.3338 ± 4 100 0.35 41 ± 2 100 0.28 15 52 ± 4 100 0.22 37 ± 4 100 0.36 42 ±5 100 0.31 30 51 ± 3 100 0.27 36 ± 5 100 0.33 35 ± 3 100 0.26 60 53 ± 2100 0.26 38 ± 4 100 0.40 41 ± 2 100 0.28 90 56 ± 5 100 0.24 37 ± 7 1000.39 40 ± 2 100 0.28 120 55 ± 5 100 0.29 38 ± 3 100 0.52 38 ± 5 100 0.27150 54 ± 2 100 0.32 39 ± 6 100 0.46 36 ± 3 100 0.25 180 54 ± 2 100 0.2738 ± 6 100 0.50 34 ± 4 100 0.25

TABLE 3 Volume particle size distribution (volume PSD) data collectedfor three separate batches for each PEG- incorporated DPPC-based SUV:PEG₆₀₀-, PEG₂₀₀₀-, and PEG₃₀₀₀-SUVs. If two or more significant (>1%)populations occur within one sample, they are reported as relativepercent populations. Errors are represented as average standarddeviations of the volume PSD. PEG₆₀₀-SUV PEG₂₀₀₀-SUV PEG₃₀₀₀-SUV VolumeVolume Volume Time PSD % PSD % PSD % (days) (nm) population PDI (nm)population PDI (nm) population PDI 0 86 ± 8 85 0.45 40 ± 4 60 0.20 60 ±3 82 0.43 612 ± 41 15 173 ± 7  40 183 ± 9  18 15 760 ± 67 74 0.56 45 ± 560 0.25 109 ± 14 70 0.41 153 ± 3  26 162 ± 8  40 543 ± 43 30 30 670 ± 4055 0.59 162 ± 12 72 0.22 86 ± 7 55 0.42 65 ± 8 45 55 ± 2 28 825 ± 57 4560 527 ± 52 82 0.40 190 ± 10 67 0.41 92 ± 8 57 0.21 98 ± 9 18 64 ± 2 33715 ± 45 43

TABLE 4 Volume particle size distribution (volume PSD) data collectedfor three separate batches for each cholesterol-incorporated DPPC-basedSUV: Chol_(5%)-, Chol_(10%)-, Chol_(15%)-, and Chol_(20%)-SUV. If two ormore significant (>1%) populations occur within one sample, they arereported as relative percent populations. Errors are represented asaverage standard deviations of the volume PSD. Chol_(5%)-SUVChol_(10%)-SUV Chol_(15%)-SUV Chol_(20%)-SUV Volume % Volume Volume %Time PSD pop- PSD % PSD % Volume pop- (days) (nm) ulation PDI (nm)population PDI (nm) population PDI PSD (nm) ulation PDI 0 68 ± 11 1000.33 80 ± 1 100 0.35 55 ± 1 100 0.28 60 ± 1 100 0.33 15 1505 ± 176 730.65  825 ± 227 100 1  710 ± 120 52 0.76  564 ± 192 95 0.88  76 ± 81 2745 ± 6 48 50 ± 6 5 30 711 ± 81 55 0.76 1511 ± 170 100 1 752 ± 95 70 13115 ± 307 100 1 54 ± 7 45 45 ± 5 30

Fourier-transformed nuclear magnetic resonance (NMR) spectroscopy ofChol-PAA was performed on a Varian INOVA-500 MHz spectrometer (Varian,Inc., Palo Alto, Calif.). Chemical shifts of ¹H NMR spectra are reportedin ppm against residual solvent resonance as the internal standard(CHCl₃=7.27 ppm, CHD₂COCD₃=2.05 ppm, CHD₂OD=3.31 ppm, D₂O=4.8 ppm.Fluorescence emission spectra were obtained on a Jobin Yvon Fluorologfluorometer (λ_(ex)=480 nm, slith width=3 nm for Doxorubicin andλ_(ex)=515 nm for Calcein).

Ultracentrifugation was carried out on a refrigerated Beckmann-CoulterOptima™ XPN ultracentrifuge (Beckmann-Coulter, Inc., Indianapolis,Ind.). Lyophilization was carried out on a Freezone lyophilizer(Labconco, Kansas City, Mo.). High-power sonication was carried outusing a titanium-alloy solid probe ultrasonicator (500 watt Vibra-Cell™VC 505, Sonics & Materials, Inc., Newtown, Conn.) set at 20 kHz, 40%intensity without pulsing. All tangential flow filtration (TFF) wasmanually carried out using a 50 kD pore size 8 cm² polystyrene (0.5 mm)MicroKros™ Module (Spectrum Labs, Rancho Dominguez, Calif.).

EXAMPLE 3 Preparation of SUV-Based Materials.

Preparation of DPPC-, DMPC-, and DOPC-SUVs. The appropriate lipid (40μmol) was dissolved in HPLC-grade chloroform (1.0 mL) in a 20 mL vialand then gently evaporated by a stream of nitrogen. The resulting lipidfilm was thoroughly dried via lyophilization for 24 hours and thenhydrated with of 20 mM HBS (5.0 mL) followed by vigorous vortexing for 5minutes. The liposome suspension was then submerged in an ice bath andprobe-sonicated for 20 minutes with the tip submerged ˜1 cm into thesample and without pulsing. The resulting suspension was then dilutedwith HBS to 10 mL and then ultracentrifuged at 104,986 g and 4° C. for 1hour. To avoid contamination by the metal filings from the probe andlarge liposomal pellet, only the top 9 mL of the supernatant wascollected using a serological pipet; this portion contains the desiredSUV dispersion.

To monitor particle size distribution as a function of storage time,three batches of each SUV were stored in 20 mL vials: the surfaces ofthe dispersions were blanketed briefly with a stream of nitrogen, thevial were capped with a plastic screw cap, and the samples were storedat 4° C. Lipid concentrations (2.45 mM for DPPC, 2.62 mM for DMPC, and4.60 mM for DOPC) were calculated using phosphorous ICP-AES.

Preparation of Polymer-Grafted Nanobins (PGNs).

In a typical preparation, Chol-PAA (10 mol % of the lipid) was dissolvedin 20 mM HBS (˜20 μL), corrected to pH 7.4 using aqueous NaOH (1 N), andadded dropwise to a stirring solution of SUVs (lipid concentrationsshown above) in a 20 mL vial. The resulting solution was allowed to stirat room temperature for 24 hours to form the desired PGN dispersion. Tomonitor particle size distribution as a function of storage time, threebatches of each PGN were stored in 20 mL vials: the surfaces of thedispersions were blanketed briefly with a stream of nitrogen, the vialswere capped with a plastic screw cap, and the samples were stored at 4°C. As the addition of Chol-PAA results in a minimal volume change, thefinal lipid concentrations of these dispersions were assumed to be thesame as those for the starting SUV dispersions (2.45 mM for DPPC, 2.62mM for DMPC, and 4.60 mM for DOPC).

Preparation of SUV-PEG600, SUV-PEG2000 and SUV-PEG3000

These SUV-PEG dispersions were prepared using the same method describedabove for the lipid-only SUVs except with different lipid formulations(90 mol % of either DPPC or DMPC and 10 mol % of either PE-PEG2000 orPE-PEG3000). There is a persistent layer of foam on the surface of thesedispersions after preparation; this layer increases in volume aftersonication. The surface of these dispersions was blanketed briefly witha stream of nitrogen, capped with a plastic screw cap, and stored underinert atmosphere at 4° C. The volume size distributions data aretabulated in Table 3. SUV-PEG600 were prepared using the same methoddescribed above for the PGN polymer insertion except that Chol-PEG600(10 mol % of the lipid concentration) was inserted into the SUVs.

Preparation of Calcein-Loaded SUVs and PGNs

This procedure is similar to the preparation of SUVs described aboveexcept that the lyophilized lipid film (40 μmol) was first hydrated withthe 20 mM calcein solution in HBS (1 mL). After vortexing for 5 minutes,this dispersion was then diluted to 5 mL with 20 mM HBS.

The resulting calcein-loaded SUVs were purified by tangential flowfiltration (TFF) during which time the particles were washed with extra20 mMHBS solution (40 mL). The resulting purified calcein-loaded SUVs(-1 mL) were used immediately for PGN preparation or stored at 4° C. forleakage evaluation. Calcein-loaded PGNs were formed using the purifiedcalcein-loaded SUVs using the same procedure described above for PGNs.

Preparation of Doxorubicin-Loaded SUVs and PGNs

In a typical experiment, DOPC (40 μmol) was dissolved in HPLC-gradechloroform in a 20 mL vial and then gently evaporated by a stream ofnitrogen. The resulting lipid film was thoroughly dried vialyophilization for 24 hours and then hydrated with of 200 mM ammoniumsulfate (5.0 mL) followed by vigorous vortexing for 5 minutes. Theliposome suspension was then submerged in an ice bath andprobe-sonicated for 20 minutes with the tip submerged ˜1 cm into thesample and without pulsing. The resulting suspension was then dilutedwith 20 mM HBS to 10 mL and then ultracentrifuged at 104,986 g and 4° C.for 1 hour. To avoid contamination by the metal filings from the probeand large liposomal pellet, only the top 9 mL of the supernatant wascollected using a serological pipet; this portion contains the desiredSUV dispersion. Using TFF the ammonium sulfate-loaded SUVs were washedwith 20 mM HBS (40 mL). The encapsulation of DXR was evaluated bycalculating the drug-to-lipid ratio which was consistently 0.22 (molDXR/ mol lipid).

Into a 20 ml vial containing a stirring solution of the ammoniumsulfate-loaded DOPC particles (5.0 mL, DOPC concentration=5.6 mM) wasslowly added an HBS solution of doxorubicin hydrochloride (17.5 mg, 1.8equiv of DOPC) over 15 minutes. The vial was then wrapped in aluminumfoil and allowed to stir at room temperature for 24 hours. Theunincorporated doxorubicin hydrochloride was separated from the loadeddispersions via gel-filtration chromatography (¼×5 inch plug ofSepharose CL-B4 and HBS elution). The purified DXR-loaded SUVs(DOPC-SUVDXR) were collected as the first ten 1 mL fractions. DXR-loadedPGN (DOPC-PGNDXR) were prepared from DOPC-SUVDXR using the same Chol-PAAincorporation procedure described above for PGN preparation.

EXAMPLE 4 Synthesis and Colloidal Stability of PGN vs SUV

SUVs with narrow size distributions were prepared from the appropriatelipid (FIG. 1) using a modification of a previously reported protocol(Wong et al. Biochemistry 1982, 21 (17), 4126-4132; herein incorporatedby reference in its entirety). To cover a broad range of phasetransition temperatures (T_(m)'s),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were selected (T_(m)=41°C., 23° C., and −21° C., respectively). The SUVs were then grafted witha cholesterol-terminated poly(acrylic acid) (Chol-PAA, M_(n)=4670 Da,PDI=0.12) following a “drop-in” method (FIG. 1) (Lee et al. J. Am. Chem.Soc. 2007, 129 (49), 15096-+; herein incorporated by reference in itsentirety). The resulting PGNs were then stored at 4° C., below theT_(m)'s of DPPC and DMPC, which enhances the fusogenicity of the SUVsderived from these lipids (Ellens et al. Biochemistry 1989, 28 (9),3692-3703; herein incorporated by reference in its entirety). Theirlong-term colloidal stabilities in 20 mM HEPES buffered saline (HBS, 150mM NaCl) were regularly monitored and compared to the unmodified SUVs(FIG. 2).

For DPPC- and DMPC-based PGN formulations (DPPC-PGN and DMPC-PGN,respectively), the dynamic light scattering (DLS, FIGS. 2a and 2b ) andtransmission electron microscopy (TEM, FIG. 3a ) data clearly indicatethat these dispersions are stable at 4° C. for at least six months afterpreparation. In stark contrast, the corresponding unmodified SUVs, whichare expected to be fusogenic at temperatures below the lipid T_(m)'s,show poor dispersability, with aggregation occurring within three weeksof preparation (FIGS. 2a, 2b, and 3b ), and formation of a visibleflocculent after four weeks (FIG. 3b inset). This is consistent withprevious observations that the r; herein incorporated by reference inits entiretyates of fusion and aggregation of SUVs are directlycorrelated with the T_(m)'s of the lipids that make up these vesicles,and these rates increase at temperatures below T_(m) (Bentz & Ellens.Colloids Surf 1988, 30 (1-2), 65-112.). While the low T_(m) of DOPC mayexplain the better dispersability of DOPC-SUVs, in comparison to DPPC-and DMPC-SUVs, when stored at 4° C., it does not fully explain theconsistently high cargo leakage rates observed for all threeformulations (FIGS. 2d, 2e, and 2f ).

EXAMPLE 5 Cargo Leakage

Sustained colloidal stability and cargo retention are key criteria forbiodelivery carriers. To evaluate the cargo-retention capability of thethree exemplary PGN formulations against the unmodified SUVs, thecarriers were loaded with the fluorescent small-molecule calcein andcompared their leakage profiles when stored at 4° C., which is therecommended storage temperature for the liposome-based drug Doxil (DoxilStorage. doxil.com/hcp/iv-preparation;

herein incorporated by reference in its entirety). Remarkably, DMPC- andDPPC-PGNs both retain over 95% of their calcein payloads over aone-month period while the corresponding SUV analogs displayedsignificant leakage (30% and 73%, respectively) (FIGS. 2d and 2e ).

Even the less fusogenic DOPC-based SUVs still leak 15% of theencapsulated calcein after one month, during which time thecorresponding DOPC-PGNs show less than 1% leakage (FIG. 2f ). Thisobservation is consistent with a previous result that showed rapidcalcein leakage occurring from egg phosphatidylcholine SUVs when thesewere stored above the lipid transition temperature (T_(m)=−10° C.),where fusion should be minimized (Mercadal et al. Biochim. Biophys.Acta, Biomembr. 1995, 1235 (2), 281-288; herein incorporated byreference in its entirety). Although the mechanism associated withleakage from cargo-bearing SUVs is currently unknown, it is likelyrelated to the high degree of membrane disorder, which has recently beencomputationally modeled; however, the embodiments described herein arenot limited to any particular mechanism of action and an understandingof the mechanism of action is not necessary to practice the embodiments.Therefore, the enhanced payload retention of the negatively charged PGNs(ζ potential=−45±3 mV) was attributed to a combination ofcholesterol-induced membrane ordering and reduction in interparticlefusion that does not exist in the unmodified, near-neutral SUVs (ζpotential=±2 mV).

In some embodiments, a biodelivery platform is capable of and/orconfigured for releasing its therapeutic payload in response to externalstimuli such as changes in biological milieu. To this end, experimentswere conducted during development of embodiments of the presentinvention to investigate the ability of PGNs to a payload of theanti-cancer drug DXR in response to in situ acidification. DXR-loadedDOPC-PGNs were incubated at 37° C. in pH 7.4 HBS for 200 minutes, thesolution was quickly acidified to pH 5.0, and regularly monitored withfluorescence spectroscopy over a period of 72 hours. Remarkably, and instark contrast to DXR-loaded DOPC-SUVs, the drug-release profile forDXR-loaded DOPC-PGNs clearly indicates a near-immediate bulk release ofDXR upon acidification to pH 5.0 (FIG. 4). It is contemplated that thisrapid release is triggered by an acid-induced phase change of thesurface bound Chol-PAA (Chen & Hoffman. Nature 1995, 373 (6509), 49-52;herein incorporated by reference in its entirety), whichcatastrophically destabilizes the high-curvature lipid membrane of thesmall-sized PGNs and causes them to rupture. This property appears to bespecific to the small size (D_(H)<50 nm) of the SUV-derived PGNs, as alarger version (D_(H)˜100 nm) releases its payloads very slowly uponacidification and requires the cross-linking of grafted PAA polymerchains into a shell around the lipid template before acid-triggeredrelease can be induced (Laaksonen et al. ChemPhysChem 2006, 7 (10),2143-2149; herein incorporated by reference in its entirety).Acidification causes the grafted Chol-PAA chains to cluster (Ringsdorfet al. Angew. Chem., Int. Ed. 1991, 30 (3), 315-318; herein incorporatedby reference in its entirety), creating large defects on the alreadyhighly curved membranes and leading to spontaneous release of thepayload (FIG. 5). Together with their small sizes, this pH-triggeredpayload release property enables SUVs to serve as “smart” therapeuticcarriers that selectively release cargo at acidic environments such asthe tumor interstitium (Smallbone et al. J. Theor. Biol. 2008, 255 (1),106-112; herein incorporated by reference in its entirety) and the lumenof late endosomes (Huotari & Helenius. EMBO J. 2011, 30 (17), 3481-3500;herein incorporated by reference in its entirety).

EXAMPLE 6 Mechanism of Chol-PAA Stabilization

To test whether charge-induced repulsion and/or the steric bulk providedby Chol-PAA grafts is the primary cause of enhanced PGN dispersabilityand improved cargo retention, a series of poly(ethylene glycol)(PEG)-grafted DPPC-SUVs were prepared using both PEGylated lipids(PE-PEG₂₀₀₀ and PE-PEG₃₀₀₀) and Chol-PEG₆₀₀. PEG-grafts were implementedas a charge-neutral polymer that closely resembles the Chol-PAA graftsof the PGN particles. PEG₂₀₀₀-grafts were specifically selected becauseit has the same degree of polymerization as the Chol-PAA, while PEG₆₀₀and PEG₃₀₀₀ were selected to provide a range of steric environments forcomparison.

Chol-PEG₆₀₀-, PE-PEG₂₀₀₀-, and PE-PEG₃₀₀₀-grafted SUVs all exhibited asteady increase in particle size over a four-week period (FIG. 6; Table3), after which rapid aggregation occurred along with visible flocculentformation. This aggregation has been previously reported for DOPC-basedSUVs (Evans & Lentz. Biochemistry 2002, 41 (4), 1241-1249; hereinincorporated by reference in its entirety) and is not surprisingconsidering the near-neutral ζ potential (±2 mV) of these PEG-graftedSUVs, following the prediction of DLVO theory. These observationssuggest that simple PEGylation of SUVs, as employed in Doxil™ to improvestabilization and engender in vivo stealth capabilities and longercirculation, will not lead to a robust ultra-small liposome carrier.

To elucidate the stabilizing effects contributed by cholesterol, whichis known to prevent SUV fusion (Mercadal et al. Biochim. Biophys. Acta,Biomembr. 1995, 1235 (2), 281-288; herein incorporated by reference inits entirety), in the Chol-PAA-stabilized PGN platform, experiments wereconducted during development of embodiments described herein to examinethe colloidal stability of DPPC-SUVs composed of 5, 10, 15, and 20 mol %of cholesterol (See Tables 4). Faster particle aggregation was observedfor all of these formulations compared to the unmodified DPPC-SUVs,which is consistent with previous reports (Roy et al. Langmuir 2010, 26(24), 18967-18975; Mcconnell & Schullery. Biochim. Biophys. Acta 1985,818 (1), 13-22; herein incorporated by reference in their entireties).Together, these experimental controls further indicate PGN stabilizationby Chol-PAA grafts is primarily conferred by charge-induced repulsionbetween particles; although the present invention is not limited to anyparticular mechanism of action and an understanding of the mechanism ofaction is not necessary to practice the present invention.

EXAMPLE 7 Methods Calcein Leakage Evaluation

To evaluate the calcein leakage as a function of storage time, threebatches of calcein-loaded PGN and SUV dispersions were stored in sealed20 mL vials under inert atmosphere at 4° C. The lipid/calcein ratio wasthen monitored directly after synthesis, after two weeks of storage, andafter a period of 1 month. At each evaluation, the calcein-loadedsamples were re-purified using TFF (same purification method asdescribed above), and the calcein/lipid ratio was revaluated as well assize measurements. The concentration of calcein in solution was measureddirectly by fluorescence spectroscopy against a calibration curve, andthe concentration of lipid was calculated from measured phosphorouscontent by ICP-OES.

Cholesterol Addition Experiments

To a stirring batch of DPPC and DMPC SUV particles, 5, 10, 15, or 20 mol% of cholesterol dissolved in 20 mM HBS was added dropwise over 15minutes. The SUV mixtures were allowed to stir at room temperature for24 hours. The volume-average particle size distribution was monitoredover several months.

Doxorubicin Release Experiments

A quartz fluorescence cuvette (1 mL) equipped with a magnetic micro-stirbar was loaded with 20 mM HBS (900 μL) and of DOPC-PGN_(DXR) (100 μL ofa 3.5 mM lipid solution in 20 mM HBS) The cuvette was placed in atemperature-controlled flourimeter mount which enabled warming to 37° C.and constant stirring during real-time measurements. The fluorescencewas measured regularly at pH 7.4 at 10-minute intervals, within thefirst 200 minutes. At the 200-minute mark, an aliquot (10 μL) of 1 MHCl, a pre-determined amount to acidify solution to pH 5.0, was added tothe cuvette and measurements continued at 10-minute intervals for thenext 100 minutes. After 720 minutes of fluorescence measurements, analiquot (50 uL) of Triton-X 100 solution (10% in water) is added and theresulting mixture was allowed to stir for five minutes. The fluorescencethat was measured represents the value for 100% release of DXR, whichwas used to normalize release values. We attribute an increase influorescence to an increased release of doxorubicin because thefluorescence of doxorubicin encapsulated within liposomes isself-quenched and is only restored upon release.^(S4)

TABLE 5 ζ potential (mV) of DPPC, DMPC, and DOPC-based PGN and SUVsamples after preparation (0 days) and after 180 days of storage at 4°C. ζ potential (mV) Time DPPC-based DMPC-based DOPC-based (days) PGN SUVPGN SUV PGN SUV 0 −37 ± 3 −4 ± 1 −43 ± 2 +1 ± 1 −45 ± 3 −2 ± 1 180 −41 ±4 −2 ± 1 −40 ± 4 +3 ± 1 −42 ± 2 −3 ± 1

All publications and patents listed below and/or provided herein arehereby incorporated by reference in their entireties. Variousmodifications and variations of the described compositions and methodsof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the relevant fields areintended to be within the scope of the present invention.

1. A composition comprising a polymer-grafted nanobin (PGN), whereinsaid PGN comprises a small unilaminer vesicle comprising aphospholipid-based bilayer with surface-exposed polymers extendingtherefrom.
 2. The composition of claim 1, wherein said small unilaminervesicle is between 15 and 50 nm in diameter.
 3. (canceled)
 4. Thecomposition of claim 1, wherein said small unilaminer vesicle comprises1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),Dipalmitoylphosphatidylcholine (DPPC), and/or1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC).
 5. The composition ofclaim 1, wherein said surface-exposed polymers comprise poly(acrylicacid) extending from cholesterol-terminated poly(acrylic acid)(Chol-PAA), wherein the cholesterol portion of the Chol-PAA is insertedinto the bilayer.
 6. (canceled)
 7. The composition of claim 1, whereinsaid PGN further comprises a molecular payload encapsulated within saidsmall unilaminar vesicle.
 8. The composition of claim 7, wherein saidmolecular payload comprises a small molecule, peptide, or nucleic acid.9. The composition of claim 7, wherein, said PGN releases less than 20%of said payload over the course of one month at normal physiologicconditions, and said PGN releases at least 50% of said payload over thecourse of less than one hour at a pH between 4.0 and 6.0.
 10. (canceled)11. The composition of claim 1, wherein said surface-exposed polymersare not cross-linked.
 12. The composition of claim 1, comprising apolymer-grafted nanobin (PGN), wherein said PGN comprises a smallphospholipid-based unilaminer vesicle (SUV) comprisingcholesterol-terminated poly(acrylic acid) groups (Chol-PAA), whereinsaid Chol-PAA is oriented such that said poly(acrylic acid) is surfaceexposed and extends from said SUV into the surrounding environment,wherein said SUV encompasses a molecular payload comprising one or morepeptides, nucleic acids, and/or small molecules.
 13. A method of drugdelivery to a low-pH microenvironment within a subject comprising: (a)administering a polymer-grafted nanobin (PGN) of claim 12 to saidsubject; and (b) allowing said PGN to migrate from physiologicconditions to an acidic microenvironment.
 14. The method of claim 13,wherein the PGN is administered locally at or near the acidicmicroenvironment.
 15. The method of claim 13, wherein the PGN isadministered systemically.
 16. The method of claim 13, wherein saidacidic microenvironment is a tumor.
 17. The method of claim 16, whereinsaid therapeutic agent is a chemotherapeutic.
 18. The method of claim16, wherein said payload is a nucleic acid-based therapy.
 19. The methodof claim 13, wherein said PGN releases less than 20% of said payloadover the course of one month at normal physiologic conditions andreleases at least 50% of said payload over the course of less than onehour at a pH between 4.0 and 6.0.
 20. The method of claim 13, whereinthe PGN is co-administered with an additional therapeutic agent.
 21. Themethod of claim 16, further comprising a step of surgically removing thetumor.
 21. (canceled) 22.-29. (canceled)
 30. A method of preparing apolymer-grafted nanobin (PGN) comprising: (a) preparing a lipid mixtureby dissolving selected lipids in an organic solvent; (b) hydrating theproduct of step (a) with an aqueous hydration solvent to form liposomes;(c) sizing the liposomes to yield small unilaminar vesicles (SUVs); and(d) incubating the SUVs with lipid-anchored polymer to generate PGNswith surface exposed polymer. 31.-46. (canceled)
 47. The method of claim21, wherein the administration step is performed after surgical removalof the tumor.